WO2008153231A1 - Procédé d'isolement de cellules vasculaires endothéliales de corps embryoïdes différenciés de cellules souches enbryonnaires - Google Patents

Procédé d'isolement de cellules vasculaires endothéliales de corps embryoïdes différenciés de cellules souches enbryonnaires Download PDF

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Publication number
WO2008153231A1
WO2008153231A1 PCT/KR2007/002840 KR2007002840W WO2008153231A1 WO 2008153231 A1 WO2008153231 A1 WO 2008153231A1 KR 2007002840 W KR2007002840 W KR 2007002840W WO 2008153231 A1 WO2008153231 A1 WO 2008153231A1
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WIPO (PCT)
Prior art keywords
cells
embryoid bodies
vascular endothelial
embryonic stem
trypsin
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PCT/KR2007/002840
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English (en)
Inventor
Hyung-Min Chung
Sung-Hwan Moon
Ju-Mi Kim
Soo-Hong Lee
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Chabiotech Co., Ltd.
College Of Medicine Pochon Cha University Industry Academic Cooperation Foundation
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Application filed by Chabiotech Co., Ltd., College Of Medicine Pochon Cha University Industry Academic Cooperation Foundation filed Critical Chabiotech Co., Ltd.
Priority to US11/913,295 priority Critical patent/US7919318B2/en
Priority to JP2010512048A priority patent/JP2010528674A/ja
Priority to PCT/KR2007/002840 priority patent/WO2008153231A1/fr
Publication of WO2008153231A1 publication Critical patent/WO2008153231A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the present invention relates to a process for isolating vascular endothelial cells from embryoid bodies differentiated from embryonic stem cells.
  • Cardiovascular diseases are one of the leading causes of death worldwide. It is estimated that every year, about 12 million people throughout the world die due to cardiovascular diseases. Cardiovascular diseases are considered to be very serious diseases that cost the United States US$ 260 billion every year, but no effective treatment has yet been identified.
  • Human embryonic stem cells retain totipotency that is the ability to differentiate into three germ cell layers (endodermal, ectodermal, mesodermal) which organize the human body. Human embryonic stem cells can be differentiated into specific cells according to their surrounding environment, and thus, are expected to become potent tools that can achieve significant progress in the medical and science fields. Thus, it is expected that studies of human embryonic stem cells can provide important clues for primitive aspects of early stages of human differentiation and can play a critical role in studies of cell therapy for cardiovascular diseases and incurable diseases, such as Parkinson's disease, myocardial infarction, diabetes, and leukemia.
  • Human embryonic stem cells can be obtained by isolating and culturing the inner cell mass of an early-stage human embryo known as "blastocyst". Human embryonic stem cells retain totipotency, and at the same time, can be maintained in an undifferentiated state and can be continuously sub-cultured (Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM, Embryonic stem cell lines derived from human blastocysts. Science (1998) 282:1145-1147). Thus, if conditions for differentiation into specific cells, proliferation, isolation, and recovery are developed and established, cell therapy studies are expected to face a major turning point.
  • human embryonic stem cells have focused on establishing differentiation conditions that can induce the differentiation of human embryonic stem cells into specific cells, such as neural cells, vascular endothelial cells, cardiac cells, endothelial cells, and hepatocytes.
  • EBs Differentiated embryoid bodies
  • EBs Differentiated embryoid bodies
  • a technique of effectively isolating only target cells after differentiation is essentially required.
  • satisfactory methods capable of selectively and efficiently isolating only target cells have not yet been reported.
  • Zhang SC et al. reported a method for isolating neural progenitor cells from differentiated embryoid bodies by dispase treatment (Zhang SC, Thomson JA et al. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nature Biotech (2001) 19, 1129-1133).
  • Zhang SC et al's report is related to isolation of neural progenitor cells and is silent about whether or not the isolation method can be applied to other types of differentiated cells, e.g., vascular endothelial cells.
  • FACS Fluorescence Activated Cell Sorter
  • trypsin and ethylenediaminetetraacetate EDTA
  • the present invention provides a process for isolating vascular endothelial cells differentiated from embryonic stem cells.
  • FIG. 1 is an image of embryoid bodies differentiated from human embryonic stem cells and an immunostained image showing the location of vascular endothelial cells in the embryoid bodies;
  • FIG. 2 is an image showing the degree of cell detachment from differentiated embryoid bodies according to the types of enzymes
  • FIG. 3 is an image showing the degree of cell detachment from embryoid bodies according to the concentration of trypsin-EDTA;
  • FIG. 4 is optical microscopic images of vascular endothelial cells according to the treatment steps of trypsin and ethylenediaminetetraacetate (EDTA);
  • FIG. 5 shows RT-PCR results for undifferentiated human embryonic stem cells, outer cells of embryoid bodies, and vascular endothelial cells isolated according to the present invention.
  • FIGS. 6 and 7 are images obtained by culturing outer cells of embryoid bodies and vascular endothelial cells isolated according to the present invention, respectively, on Matrigels.
  • vascular endothelial cells refers to cells which form an inner layer of a blood vessel and express markers such as PECAM, CD34, VE cadherin, eNOs, etc.
  • embryoid bodies refers to aggregates composed of three germ cell layers (endodermal, ectodermal, mesodermal) differentiated from human embryonic stem cells. Embryoid bodies can be maintained in an appropriate medium.
  • the present inventors investigated the distribution of vascular endothelial cells in differentiated embryoid bodies using anti-PECAM antibodies, and as a result, found that most vascular endothelial cells are present in the center regions of embryoid bodies (see FIG. 1).
  • embryoid bodies are subjected to a two-step trypsin-EDTA treatment including a lower dose trypsin-EDTA treatment and a higher dose trypsin-EDTA treatment.
  • vascular endothelial cells When the embryoid bodies are treated with a lower dose trypsin-EDTA, the outer regions of the embryoid bodies containing only a trace amount of vascular endothelial cells are effectively removed and regions of the embryoid bodies containing large amounts of vascular endothelial cells are kept intact.
  • EDTA-trypsin When the embryoid bodies are treated with a higher dose EDTA-trypsin, vascular endothelial cell clusters are separated into single cells. As a result, vascular endothelial cells can be simply recovered in a high yield of about 30% or more.
  • the isolation method of the present invention includes forming embryoid bodies by culturing embryonic stem cells.
  • the embryonic stem cells comprehend all embryonic stem cells derived from mammals.
  • the embryonic stem cells may be embryonic stem cells derived from human.
  • the term "human embryonic stem cells” refers to totipotent cells derived from the inner cell mass of human morula.
  • the human embryonic stem cells may be, but not limited to, CHA-hES3 (Ahn SE 1 Kim S, Park KH, Moon SH, Lee HJ, Kim GJ, Lee YJ, Park KH, Cha KY, Chung HM.
  • Primary bone-derived cells induce osteogenic differentiation without exogenous factors in human embryonic stem cells. Biochem Biophys Res Commun.
  • the human embryonic stem cells can be easily established by those of ordinary skill in the art. Formation of embryoid bodies from human embryonic stem cells can be performed by a method commonly known in the art. For example, according to the method disclosed in Levenberg S, Golub JS, Amit M, Itskovitz-Eldor J, Langer R. PNAS (2002) 99, 4391-4396, embryoid bodies can be formed by culturing human embryonic stem cells in a DMEM/F12 medium supplemented with serum (or serum replacement), L-glutamine, nonessential amino acid, and ⁇ -mercaptoethanol.
  • the embryoid bodies may be used in the form of a culture medium obtained by suspension-culturing them in an EB culture medium comprising serum replacement, mercaptoethanol, nonessential amino acid, and 80% KO-DMEM (KNOCKOUT Dulbecco's modified Eagle's medium) for about 7-10 days.
  • the suspension-cultured embryoid bodies are cultured in a culture dish including a DMEM supplemented with fetal bovine serum (FBS), mercaptoethanol, and nonessential amino acid for about 24 hours so that attached embryoid bodies spread out.
  • FBS fetal bovine serum
  • trypsin may be a trypsin derived from a mammal (e.g., porcine trypsin) or a recombinant trypsin obtained by a recombination technique.
  • EDTA used in the isolation method of the present invention may be in the form of ethylenediaminetetraacetic acid, ethylenediaminetetraacetate disodium, or ethylenediaminetetraacetate disodium dihydrate.
  • EDTA may be in the form of ethylenediaminetetraacetate disodium dihydrate.
  • Trypsin and EDTA may be used in the form of a solution in sterile physiological saline, preferably in an about 0.9% sodium chloride solution.
  • a commercially available trypsin-EDTA solution (Sigma, U.S.A.) may be used in the form of a dilute solution containing desired concentrations of trypsin and EDTA.
  • the concentration of trypsin is 0.005 - 0.015%, preferably about 0.01%, and the concentration of EDTA is 0.05 - 0.15 mM, preferably about 0.1 mM.
  • Trypsin and EDTA can be removed by washing with a physiologically compatible buffer, e.g., a phosphate buffered saline, or a medium.
  • a physiologically compatible buffer e.g., a phosphate buffered saline, or a medium.
  • step (b) vascular endothelial cell clusters obtained in step (a), i.e., embryoid bodies containing large amounts of vascular endothelial cells are separated into single cells.
  • concentration of trypsin is 0.1 - 0.5 %, preferably about 0.25%
  • concentration of EDTA is
  • the trypsin-EDTA treatment may be performed for 3 to 10 minutes, preferably about 5 minutes.
  • vascular endothelial cells when isolating vascular endothelial cells by a two-step trypsin-EDTA treatment including a lower dose trypsin-EDTA treatment and a higher dose trypsin-EDTA treatment, only regions of embryoid bodies containing a trace amount of vascular endothelial cells differentiated from human embryonic stem cells are selectively and simply removed, thereby minimizing cell damage, and thus, keeping the intrinsic characteristics of vascular endothelial cells intact. Therefore, obtained cells can maintain good blood vessel-forming capabilities.
  • Example 1 Mouse fibroblasts (STO cells, 2.5 x 10 5 cells/well), which had been treated with mitomycin-C for two hours to prevent cell proliferation, and feeder cell culture media (90% DMEM supplemented with 10% FBS, 0.1 mM mercaptoethanol, and 1 % nonessential amino acid (Gibco)) were plated on gelatin-coated culture dishes, and the STO cells were cultured for 24 hours. The culture dishes were washed twice with phosphate buffered saline media.
  • feeder cell culture media 90% DMEM supplemented with 10% FBS, 0.1 mM mercaptoethanol, and 1 % nonessential amino acid (Gibco)
  • the media in the culture dishes were replaced with culture media (80% KO-DMEM supplemented with 20% serum replacement (SR), 0.1 mM mercaptoethanol, 1% nonessential amino acid (Gibco), and 4 ng/ml bFGF), and human embryonic stem cells (CHA-hES3) were cultured for about seven days.
  • Human embryonic stem cell colonies formed in the culture media were detached from neighboring feeder cells by a glass pipette, and were then suspension-cultured in culture media including 20% serum replacement (Gibco), 0.1 mM mercaptoethanol, 1% nonessential amino acid (Gibco), and 80% KO-DMEM for about 10 days to form embryoid bodies.
  • the suspension-cultured embryoid bodies were transferred to culture dishes including culture media (90% DMEM supplemented with 10% FBS, 0.1 mM mercaptoethanol, and 1% nonessential amino acid (Gibco)), and were then cultured for 24 hours so that the embryoid bodies spread out. Then, the media in the culture dishes were replaced with fresh media for about 10 days, every other day.
  • the distribution of vascular endothelial cells in differentiated embryoid bodies was investigated using anti-PECAM antibodies and the results are shown in FIG. 1. As shown in FIG. 1, most vascular endothelial cells are present in the center regions of embryoid bodies.
  • Step 1 lower dose trvpsin-EDTA treatment
  • the embryoid bodies obtained in Example 1 were seeded on culture media (90% DMEM supplemented with 10% FBS, 0.1 mM mercaptoethanol, and 1% nonessential amino acid (Gibco)).
  • a 10-fold dilute solution of a trypsin-EDTA solution (Sigma, U.S.A.) of 0.25% trypsin and 1 mM EDTA in a 0.9% sodium chloride solution was added to the culture media, and the embryoid bodies were cultured for five minutes.
  • Step 2 separation into single cells
  • the embryoid bodies obtained in step 1 were washed with a phosphate buffered saline to thereby completely remove cell clusters in the outer regions of the suspended embryoid bodies.
  • a trypsin-EDTA solution (Sigma, U.S.A.) of 0.25% trypsin and 1 mM EDTA in a 0.9% sodium chloride solution was added, and the remaining cell clusters were cultured for five minutes to thereby separate the cell clusters into single cells.
  • Example 3 The same treatment as in Example 2 was performed except that in step 1 , a 8-fold dilute solution of the trypsin-EDTA solution (Sigma, U.S.A.) was added to embryoid bodies, and the embryoid bodies were cultured for one, three, and five minutes.
  • a 8-fold dilute solution of the trypsin-EDTA solution Sigma, U.S.A.
  • Example 4 The same treatment as in Example 2 was performed except that in step 1 , a 6-fold dilute solution of the trypsin-EDTA solution (Sigma, U.S.A.) was added to embryoid bodies, and the embryoid bodies were cultured for one, three, and five minutes.
  • a 6-fold dilute solution of the trypsin-EDTA solution Sigma, U.S.A.
  • Example 2 The embryoid bodies obtained in Example 1 were seeded on culture media (90% DMEM supplemented with 10% FBS, 0.1 mM mercaptoethanol, and 1% nonessential amino acid (Gibco)). Then, dispase (Comparative Example 1), collagenase (Comparative Example 2), and a 1X cell dissociation buffer (Comparative Example 3) were added to the culture media, and the embryoid bodies were cultured for 20 minutes.
  • culture media 90% DMEM supplemented with 10% FBS, 0.1 mM mercaptoethanol, and 1% nonessential amino acid (Gibco)
  • dispase Comparative Example 1
  • collagenase Comparative Example 2
  • a 1X cell dissociation buffer Comparative Example 3
  • step 1 5- and 2-fold dilute solutions of the trypsin-EDTA solution (Sigma,
  • Example 2 The embryoid bodies obtained in Example 1 were treated with dispase, collagenase, a cell dissociation buffer, and trypsin-EDTA, like in Comparative Examples 1-3, and the degree of cell detachment from the embryoid bodies were measured using an optical microscope. The results are shown in FIG. 2.
  • RT-PCR was performed for the undifferentiated human embryonic stem cells used in Example 1 , and the outer and inner cells of the embryoid bodies obtained in step 1 of Example 2, and PECAM specific to vascular endothelial cells was quantified. The results are shown in FIG.
  • embryoid bodies when embryoid bodies are treated according to the present invention, outer cells of the embryoid bodies containing a trace amount of vascular endothelial cells can be effectively removed, and the embryoid bodies can be simply separated into single cells.
  • the recovery rate of vascular endothelial cells was about 30% or more.
  • the viability of the cells obtained in Example 2 was tested with trypan blue staining. As a result, it was determined that 95% or more of the cells were alive.
  • Example 4 RT-PCR was performed for the undifferentiated human embryonic stem cells used in Example 1 , and the outer and inner cells of the embryoid bodies obtained in step 1 of Example 2, according to an Asikainen TM et al's method (Enhancement of angiogenic effectors through hypoxia-inducible factor in preterm primate lung in vivo. Am J Physiol Lung Cell MoI Physiol. 2006 May 5), and quantitative analysis for vascular endothelial cells was performed. The results are shown in FIG. 5. As can be seen from FIG.
  • the proportion of vascular endothelial cells among the inner cells of the embryoid bodies is about 7-fold or more higher than the proportion of vascular endothelial cells among the outer cells of the embryoid bodies.
  • Example 6 the outer cells of the embryoid bodies obtained in Example 1 and the single cells obtained in Example 2 were cultured on Matrigels, and the results are shown in FIGs. 6 and 7, respectively.
  • FIGs. 6 and 7 cells isolated according to the present invention exhibit blood vessel-forming capabilities which are similar to those of vascular endothelial cells.
  • an isolation method of the present invention among embryoid bodies differentiated from human embryonic stem cells, only regions of the embryonic bodies containing a trace amount of vascular endothelial cells are selectively and easily removed, thereby enabling rapid and efficient recovery of differentiated vascular endothelial cells. Due to the use of a low-concentration enzyme solution, cell damage is minimized, and thus, blood vessel-forming capabilities which are the intrinsic characteristics of vascular endothelial cells can be maintained at high level.

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Abstract

L'invention porte sur un procédé permettant d'isoler des cellules endothéiale vasculaires de corps embryoïdes différenciés à partir de cellules souches embryonnaires, qui consiste à; (a) traiter lesdits corps embryoïdes différenciés de cellules souches embryonnaires avec 0,005 - 0,015 % de trypsine et 0,05 - 0,15 mM d'éthylènediaminetétraacétate (EDTA) pour obtenir des amas de cellules endothéliales vasculaires; et (b) traiter les amas de cellules endothéliales vasculaires avec 0,1 - 0,5 % de trypsine et 0,5 - 2 mM de EDTA pour dissocier lesdits amas, en cellules individuelles.
PCT/KR2007/002840 2007-06-13 2007-06-13 Procédé d'isolement de cellules vasculaires endothéliales de corps embryoïdes différenciés de cellules souches enbryonnaires WO2008153231A1 (fr)

Priority Applications (3)

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US11/913,295 US7919318B2 (en) 2007-06-13 2007-06-13 Process for isolating vascular endothelial cells from embryoid bodies differentiated from embryonc stem cells
JP2010512048A JP2010528674A (ja) 2007-06-13 2007-06-13 胚性幹細胞から分化した胚様体から血管内皮細胞を単離する方法
PCT/KR2007/002840 WO2008153231A1 (fr) 2007-06-13 2007-06-13 Procédé d'isolement de cellules vasculaires endothéliales de corps embryoïdes différenciés de cellules souches enbryonnaires

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PCT/KR2007/002840 WO2008153231A1 (fr) 2007-06-13 2007-06-13 Procédé d'isolement de cellules vasculaires endothéliales de corps embryoïdes différenciés de cellules souches enbryonnaires

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US20060199263A1 (en) * 2003-06-27 2006-09-07 Auger Francois A Method of isolating cells from umbilical cord
KR100744440B1 (ko) * 2006-06-01 2007-08-01 (주) 차바이오텍 배아 줄기 세포로부터 분화된 배상체로부터 혈관 내피세포의 분리방법

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JP2003009854A (ja) * 2001-04-09 2003-01-14 Kyowa Hakko Kogyo Co Ltd エンブリオイドボディ形成方法及びその用途
US20040009589A1 (en) * 2002-03-26 2004-01-15 Shulamit Levenberg Endothelial cells derived from human embryonic stem cells
WO2005105984A2 (fr) * 2004-04-12 2005-11-10 The Trustees Of The University Of Pennsylvania Conditions de culture et facteurs de croissance modifiant une determination de devenir, autorenouvellement et dilatation de cellules souches spermatogoniales de souris
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US20040096967A1 (en) * 2002-10-31 2004-05-20 Pfizer Inc Suspension method for producing embryoid bodies, compositions and methods related thereto
US20060199263A1 (en) * 2003-06-27 2006-09-07 Auger Francois A Method of isolating cells from umbilical cord
KR100744440B1 (ko) * 2006-06-01 2007-08-01 (주) 차바이오텍 배아 줄기 세포로부터 분화된 배상체로부터 혈관 내피세포의 분리방법

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JP2010528674A (ja) 2010-08-26
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